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目的分离甲型H1N1流感病毒,分析福建省首例病毒分离株全基因组序列和遗传特征,为研究病毒进化、致病性、流行规律提供科学依据。方法采用MDCK细胞和Real-time PCR法进行病毒分离、鉴定;提取病毒RNA,通过RT-PCR扩增其8个基因片段,测定核苷酸序列,利用生物信息软件拼接全基因组序列;分析重要基因位点,利用GENBANK中相关序列对首例病毒分离株A/Fujian/01/2009(H1N1)进行基因进化树分析。结果从82例甲型H1N1流感确诊病例标本中分离出50株甲型H1N1流感病毒,第一代分离阳性率60.98%。在福建省首次获得甲型H1N1流感病毒株及全基因组序列。基因组序列分析证明:该毒株与2009年大流行株高度同源,其基因组存在四源重组现象;氨基酸位点分析其对达菲药物敏感,对金刚烷胺类药物耐药;相对于猪流感代表株A/Swine/Iowa/15/1930(H1N1)存在6个HA抗原决定簇位点变异。结论MD-CK细胞对甲型H1N1流感病毒具有较高敏感性;福建省首例甲型H1N1流感病例分离病毒株与北美流行株高度同源;相对于以往古典型猪流感代表株出现了HA蛋白抗原性漂移;为今后进一步开展甲型H1N1流感病毒分子生物学研究奠定基础。
Objective To isolate Influenza A (H1N1) virus and analyze the whole genome sequence and genetic characteristics of the first isolate in Fujian Province, providing a scientific basis for studying the evolution, pathogenicity and prevalence of the virus. Methods The virus was isolated and identified by MDCK cells and Real-time PCR. The viral RNA was extracted and the 8 gene fragments were amplified by RT-PCR. The nucleotide sequences were determined. The whole genome sequence was spliced by bioinformatics software. The first virus isolate A / Fujian / 01/2009 (H1N1) was analyzed by phylogenetic tree using the related sequences in GENBANK. Results A total of 50 influenza A (H1N1) viruses were isolated from 82 confirmed cases of Influenza A (H1N1). The positive rate of the first generation was 60.98%. In Fujian Province for the first time to get type A H1N1 influenza virus strains and whole genome sequence. Genomic sequence analysis showed that the strain was highly homologous to the 2009 pandemic strain and its genome had a four-source recombination phenomenon. Amino acid site analysis was sensitive to Tamiflu and was resistant to amantadine. Compared with the swine flu The representative strain A / Swine / Iowa / 15/1930 (H1N1) has 6 HA epitope variation. Conclusion MD-CK cells are highly sensitive to influenza A (H1N1) virus. The first case of influenza A (H1N1) isolates from Fujian Province is highly homologous to the North American strain. Compared with the classical strain of classical swine influenza, HA protein Antigenic drift; and lay the foundation for the further study of the molecular biology of Influenza A (H1N1) virus in the future.